Date Published: September 13, 2018
Publisher: Public Library of Science
Author(s): Yanni Yin, Sisi Wu, Chaonan Chui, Tianling Ma, Huixian Jiang, Matthias Hahn, Zhonghua Ma, Yuanchao Wang.
The mitogen-activated protein kinase (MAPK) cassette of the cell wall integrity (CWI) pathway is primarily responsible for orchestrating changes of cell wall. However, functions of this cassette in other cellular processes are not well understood. Here, we found that the Botrytis cinerea mutant of MAPK kinase (BcMkk1) displays more serious defects in mycelial growth, conidiation, responses to cell wall and oxidative stresses, but possesses less reduced virulence than the mutants of its upstream (BcBck1) and downstream (BcBmp3) kinases. Interestingly, BcMkk1, but not BcBck1 and BcBmp3, negatively regulates production of oxalic acid (OA) and activity of extracellular hydrolases (EHs) that are proposed to be virulence factors of B. cinerea. Moreover, we obtained evidence that BcMkk1 negatively controls OA production via impeding phosphorylation of the Per-Arnt-Sim (PAS) kinase BcRim15 by the Ser/Thr kinase BcSch9. In addition, the fungal Pro40 homolog BcPro40 was found to interact simultaneously with three MAPKs, implying that BcPro40 is a scaffold protein of the CWI pathway in B. cinerea. Taken together, results of this study reveal that BcMkk1 negatively modulates virulence via suppressing OA biosynthesis in B. cinerea, which provides novel insight into conserved and species-specific functions of the MAPK kinase in fungi.
The cell wall integrity (CWI) signaling pathway is required for remodeling of the fungal cell wall in a highly regulated and polarized manner during growth, morphogenesis, and responses to environmental challenges . This pathway has been well understood in Saccharomyces cerevisiae [2–4]. In the yeast, cell surface sensors Wsc1, -2, -3, Mid2 and Mtl1 respond to cell wall stress stimuli by activating the small G protein Rho1 via its guanosine nucleotide exchange factor Rom2. Rho1 then (interacts with and) activates Pkc1, which in turn triggers a mitogen-activated protein kinase (MAPK) cascade consisting of the MEKK Bck1, a pair of redundant MEKs (Mkk1 and Mkk2) and Mpk1, also named Slt2. Activation of the MAPK cascade transmits signals to several transcription factors including Swi4, Swi6 and Rlm1, which activate expression of genes involved either in cell wall biosynthesis and remodeling or in cell cycle progression [2–4].
The conserved CWI pathway is responsible for the integrity of cell wall in many fungal species [11,12,62]. Similar to the reports from other fungi, the MAPK mutants in the CWI pathway of B. cinerea showed increased sensitivity to CR, which destroys the connection of chitin microfibril to β-glucan . qRT-PCR assays revealed that chitin synthase-encoding genes were dramatically down-regulated in the B. cinerea MAPK mutants (S5 Fig). Previous studies have shown that the three MAPKs Bck1, Mkk1 and Slt2 function clearly in the same way in S. cerevisiae, A. fumigatus, M. oryzae, Ustilago maydis, Ashbya gossypii and S. macrospora [12,14,64–66]. Moreover, the three MAPK mutants of F. graminearum or M. oryzae showed similar morphological characters (S6A Fig). However, in this study, ΔBcMkk1 exhibited more serious defects in mycelial growth, conidiation and responses to cell wall and oxidative stresses than ΔBcBck1 and ΔBcBmp3 (Figs 1 and 2), although western blotting and Y2H assays confirmed the three kinases belong to the CWI pathway of B. cinerea (Figs 2F and 6B). Interestingly, ΔBcMkk1 exhibited higher virulence than ΔBcBck1 and ΔBcBmp3 although deletion of BcBCK1, BcMKK1 or BcBMP3 led to dramatically decreased virulence on various plants as compared to the wild type. In most pathogenic fungi, the lack of CWI kinases led to decreased fungal virulence [6,9–11,66,67]. However, the negative functions of a MAPK kinase in fungal virulence has not been reported previously. Here, BcMkk1 not only positively regulates virulence resulting from controlling growth, conidiation and the responses to oxidative and cell wall stresses, but negatively modulates virulence via suppressing oxalic acid production and pathogenicity-related hydrolase activity (Fig 7). These results strongly indicated in addition to involvement in the CWI pathway, BcMkk1 carries additional functions in regulating virulence in B. cinerea.